Paradoxical attenuation of neuroinflammatory response upon LPS challenge in miR-146b deficient mice

The miR-146 family consists of two microRNAs (miRNAs), miR-146a and miR-146b (miR-146a/b), both of which are known to suppress immune responses in a variety of conditions. Here, we studied how constitutive deficiency of miR-146b (Mir146b-/-) affects lipopolysaccharide (LPS)-induced neuroinflammation in mice. Our experiments demonstrated that miR-146b deficiency results in the attenuation of LPS-induced neuroinflammation, as it was evidenced by the reduction of sickness behavior, a decrease in the inflammatory status of microglia, and the loss of morphological signs of microglial activation in the hippocampus. Gene expression analysis revealed that LPS-induced upregulation of hippocampal pro-inflammatory cytokines is attenuated in Mir146b-/- mice, compared to wild-type (WT) mice. In addition, reduced expression of the NF-κB nuclear protein p65, reduced miR-146 family target TLR4 expression and relatively stronger upregulation of miR-146a was found in Mir146b-/- mice as compared to WT mice upon LPS challenge. Compensatory upregulation of miR-146a can explain the attenuation of the LPS-induced neuroinflammation. This was supported by experiments conducted with miR-146a/b deficient mice (Mir146a/b-/-), which demonstrated that additional deletion of the miR-146a led to the restoration of LPS-induced sickness behavior and proinflammatory cytokines. Our experiments also showed that the observed upregulation of miR-146a in Mir146b-/- mice is due to the overexpression of a miR-146a transcription inducer, interferon regulatory factor 7 (Irf7). Altogether, our results show the existence of crosstalk between miR-146a and mir-146b in the regulation of LPS-induced neuroinflammation.

The miR-146 family consists of two microRNAs (miRNAs), miR-146a and miR-146b (miR-146a/b), both of which are known to suppress immune responses in a variety of conditions. Here, we studied how constitutive deficiency of miR-146b (Mir146b-/-) affects lipopolysaccharide (LPS)-induced neuroinflammation in mice. Our experiments demonstrated that miR-146b deficiency results in the attenuation of LPS-induced neuroinflammation, as it was evidenced by the reduction of sickness behavior, a decrease in the inflammatory status of microglia, and the loss of morphological signs of microglial activation in the hippocampus. Gene expression analysis revealed that LPS-induced upregulation of hippocampal pro-inflammatory cytokines is attenuated in Mir146b-/-mice, compared to wild-type (WT) mice. In addition, reduced expression of the NF-kB nuclear protein p65, reduced miR-146 family target TLR4 expression and relatively stronger upregulation of miR-146a was found in Mir146b-/-mice as compared to WT mice upon LPS challenge. Compensatory upregulation of miR-146a can explain the attenuation of the LPS-induced neuroinflammation. This was supported by experiments conducted with miR-146a/b deficient mice (Mir146a/b-/-), which demonstrated that additional deletion of the miR-146a led to the restoration of LPS-induced sickness behavior and proinflammatory cytokines. Our experiments also showed that the observed upregulation of miR-146a in Mir146b-/-mice is due to the overexpression of a miR-146a transcription inducer, interferon regulatory factor 7 (Irf7). Altogether, our results show the existence of crosstalk b e t w e e n m i R -1 4 6 a a n d m i r -1 4 6 b i n t h e r e g u l a t i o n o f L P Sinduced neuroinflammation. KEYWORDS miRNAs, miR-146b deficiency, attenuated neuroinflammation, miR-146a upregulation, miR-146a/b crosstalk Introduction Neuroinflammation is considered as an important contributing factor in various central nervous system disorders, including neuropsychiatric disorders and neurodegenerative diseases, such as major depression, schizophrenia, Alzheimer's disease, and Parkinson's disease (1)(2)(3)(4)(5). During inflammation, the termination phase is crucial to prevent tissue damage. For that, effective resolution programs are needed to prevent the progression of persistent inflammation (6). In this regard, several microRNAs (miRNAs) have been demonstrated to have a critical role in controlling inflammation (7). miRNAs are endogenous, small non-coding RNAs with ∼22 nucleotides in length, which regulate gene expression via imperfect base-pairing with the 3′-untranslated regions (3′-UTR) of multiple target mRNAs and thereby inhibit mRNA and protein expression (8). Individual miRNAs can affect different aspects of innate as well as adaptive immune cell development and functions (9). Several miRNAs have emerged as regulators of neuroinflammation, among which miR-155 has been shown to promote (10) and the miR-146 family to suppress neuroinflammation (11,12).
The miR-146 family of miRNAs consists of two members, miR-146a and miR-146b (miR-146a/b), which differ only in two nucleotides and are encoded by two distinct genes located on chromosomes 5 and 10, in the human genome and on chromosome 11 and 19 in the mouse genome, respectively (13). As miR-146a/b have identical seed sequences, they can probably target the same or very similar set of genes (13,14). Both miR-146a/b have been shown to negatively regulate the nuclear factor kappa B (NF-kB) pathway through multiple targets such as the TNF receptor-associated factor 6 (TRAF6) and interleukin-1 receptor-associated kinase 1 (IRAK1) (15). Correspondingly, miR-146a/b have been shown to suppress innate immune responses in cell culture conditions (16) and in vivo in mice, as demonstrated by the exaggerated inflammatory response of miR-146a knockout mice upon administration of lipopolysaccharide (LPS) (17,18). The lack of miR-146a or miR-146a/b also leads to changes in adaptive immune responses, as more prominent T helper (Th)1/Th17 responses have been revealed in these mice in different disease models (19)(20)(21).
In contrast to miR-146a, very few studies have addressed miR-146b function in the regulation of neuroinflammation in the central nervous system. Overexpression of miR-146b was shown to inhibit inflammatory responses via suppression of the activation of the NF-kB signaling in the brain in rat encephalopathy models (30). Another study showed that treatment of microglial cells with LPS led to increased expression of miR-146b but not miR-146a, while transfection of miR-146b into microglial cells significantly decreased LPSinduced microglial activation and downregulated IRAK1 (31). Intriguingly, we reported that constitutive miR-146b deficiency didn't cause microglial activation or signs of neuroinflammation in the hippocampus of the mouse brain (22). However, knowledge on the regulation of neuroinflammation in the absence of miR-146b is still lacking.
Hereby, we used Mir146b-/-mice to study the role of miR-146b in neuroinflammation upon LPS challenge. Interestingly, we observed blunted neuroinflammatory response in miR-146b deficient mice subjected to LPS treatment which may be due to the elevated miR-146a level, causing downregulation of inflammatory factors.

Materials and methods
Animals Two-three months old male C57BL/6J WT, Mir146b-/-and Mir146a/b-/-were housed and bred in laboratory animal facility at the Institute of Biomedicine and Translational Medicine, University of Tartu under standard conditions as previously described in (14). Mir146b-/-and corresponding WT mice used for this study were obtained by crossing Mir146b+/heterozygous mice. The Mir146a/b-/-mouse line was created by crossing Mir146b-/-and Mir146a-/-lines. The generated animals were genotyped using the following primer sequence: 146b locus 5′ forward primer-5′ CTCACACTCTTGTT CTTACCCAGTTCTT 3′; 146b locus 3′ reverse primer-5′ CAAACAAACAAACAAAAGGTTCAGCTAAG 3′; 146b locus internal reverse primer-5′ACACACAGGGCATATG A GA T CAG T T GG T T 3 ′ ; 1 4 6 a f or w a r d p r i m e r -5' ACCAGCAGTCCTCTTGATGC 3'; 146a reverse primer-3' GACGAGCTGCTTCAAGTTCC 5' and same generation littermates were used in experiments. All experiments were undertaken in agreement with the guidelines established in the Principles of Laboratory Animal Care (Directive 2010/63/EU) and the mice were group-housed with a 12h light/dark cycle with food and water available ad libitum. The Animal Experimentation Committee at the Estonian Ministry of Agriculture (no. 183 and 158, 2021) approved the experimental protocol.

Experimental plan
Male WT (n = 40) and Mir146b-/-mice (n = 44), were randomly assigned to different experimental groups. The first cohort (WT n = 12, Mir146b-/-n = 13) was used to measure bodyweight, locomotor activity and sickness behavior after LPS or saline administration during the 20h period and animals were sacrificed at 24h. This cohort was used to determine LPSinduced genotypic differences in microglial profile by flow cytometry. The second cohort (WT n = 12, Mir146b-/-n = 13) was used for gene expression analysis, miRNA quantification and Western blot, besides behaviors. The third cohort (WT n = 10, Mir146b-/-n = 12) was used for morphological analysis of ionized calcium binding adaptor molecule 1 (Iba-1) positive cells by immunohistochemistry. Fourth cohort (WT n = 6, Mir146b-/-n = 6) was used for isolation of microglial cells after LPS administration. In addition, Male WT (n = 10) and Mir146a/b-/-mice (n = 10) was used to measure sickness behavior after LPS or saline administration followed by gene expression analysis. The sequence of the experiments performed is summarized in Figure 1.

LPS treatment
LPS (derived from E. coli serotype O55:B5; Sigma-Aldrich, St. Louis, MO, USA) was dissolved in 0.9% NaCl (saline). In each animal cohort, one group was injected intraperitoneally (i.p.) with 1.5 mg/kg bodyweight of LPS solution and another group received injection of an equal volume of saline as controls. For isolated microglial cells from WT mice, 1 mg/mL of LPS for 4 h was used for stimulation.
Body weight measurement and assessment of locomotor activity Bodyweight was measured before and 24 h post LPS or saline injection in WT and Mir146b-/-mice. The effect of LPS on locomotor activity was monitored for 60 or 30 min by using PhenoTyper apparatus (Noldus, Leesburg, VA) with Ethovision XT live videotracking software (Version 8.0, Noldus Information Technologies, Leesburg, VA). Total distance traveled in the whole arena was measured.

Tail suspension test
Tail suspension test was performed as previously described (32). In separate compartments of the wooden test apparatus, each mouse was suspended by the tail to a wooden beam with an adhesive tape. The total duration of immobility time during the 6 min testing period was measured manually by using stopwatch. Immobility was defined as a complete lack of movement other than respiration, small movements of forefeet and swinging caused by earlier movements.

Flow cytometry
Flow cytometry was conducted according to the validated method published previously (33,34). Mice were euthanized with CO2 and the brain tissues were dissected out. Isolation of cells were made by homogenizing the tissues through 70 µm cell strainers (#352350, BD Bioscience) in ice-cold flow buffer (PBS with 1% fetal calf serum). Cells were blocked with 10% rat serum in ice-cold PBS for 1 h and stained with 0.  Schematic overview of the experimental plan.

Immunohistochemistry of Iba1
After 24 h of LPS administration, animals were deeply anesthetized with chloral hydrate (300 mg/kg, i.p.) and transcardially perfused using 0.9% saline and then with 4% paraformaldehyde (PFA) in phosphate buffered saline (PBS, pH = 7.4). The brains were kept in PFA solution for 72 h to reduce unspecific binding to endogenous biotin and 40 µm-thick sections were cut on a Leica VT1000S vibro-microtome (Leica Microsystems Pvt Ltd., Wetzlar, Germany) and stored at −20°C in the cryo-protectant (30% ethylene glycol, 30% glycerol in PBS: pH 7.4).
Before staining, the sections were washed three times in PBS and treated with 2% H2O2 solution for 20 min followed by incubation in 0.01 M citrate buffer (pH 6.0) at 85°C for 30 min in water bath and then stood for 30 min at room temperature. Another wash was done in PBS containing 0.1% Triton X-100. Blocking was done with solution containing 5% goat serum, 0.5% Tween-20, 0.25% Triton X-100 in 100 mM PBS for 1 h. Iba1 primary antibody (1: 700, rabbit anti-Iba1, CAF6806, FUJIFILM Wako Chemicals Europe GmbH, Neuss, Germany) was added in blocking buffer for 72 h. After being washed three times, the sections were incubated with secondary antibody (1:700, affinity purified goat anti-rabbit biotinylated IgG (H +L), Vector Laboratories) in blocking buffer at room temperature for 1 h. Iba1-positive cells were visualized using peroxidase method (ABC system and diaminobenzidine as chromogen, Vector Laboratories). The sections were dried, cleared with xylol and cover-slipped with mounting medium (Vector Laboratories, Newark, CA, USA). To control unspecific binding, some sections were used as negative controls, where incubation step with the primary antibody was omitted.
The morphological characteristics of Iba1+ microglia (cell size, cell body size, size dendritic processes), were analyzed using image analysis software (ImageJ 1.48v, http://imagej.nih.gov/ij), an algorithm previously described (35) was used. Briefly, images were converted into binarized 8-bit format and "adjusted threshold" and "analyze particles" functions were used to apply intensity thresholds and size filter. To measure the total cell size, the threshold was maintained at the level that was automatically provided by the ImageJ program, and size filter of 150 pixels was applied. To measure the total cell body size, the threshold was lowered by 40 points and no size filter was applied. The counts of Iba-1 positive cells were obtained from images according to the algorithm described previously (36) using "analyze particles" command in ImageJ software.

Isolation of microglia
Brain cells were isolated as previously described (37). Tissues were mechanically homogenized and passed through a 70 mm nylon cell strainer (352350, BD Bioscience) with approximately 10-15 ml of 1X DPBS supplemented with 0.2% glucose into a 50 ml conical tube. Isotonic percoll dilutions were made by diluting stock percoll (GE-healthcare, 17-0891-01, Chicago, IL, USA) at a 9:1 ratio with 10X PBS to make stock isotonic percoll (SIP), which is considered 100% SIP. The layers of percoll were created by diluting the 100% SIP with 1X Dulbecco's phosphate buffered saline (DPBS) to make 70% SIP, 50% SIP and 35% SIP. Obtained homogenate was then centrifuged at 600× g for 6 min at room temperature. Supernatant was decanted and the pellet resuspended in 6 ml of the 70% SIP. Resuspended homogenate was transferred to a 15 ml tube and 3 ml of the 50% SIP was carefully layered over. Another 3 ml of the 35% SIP was carefully layered on top of the 50% SIP layer, and 2 ml of 1X DPBS was layered on top of the 35% layer. The prepared 15ml tubes were then centrifuged at 2000× g for 20 min at room temperature without brake. Three discrete layers were established after centrifugation. Microglial cells were collected from the interface between the 50-70% SIP. Isolated microglial cells were resuspended in sterile 1X DPBS and centrifuged at 600× g for 6 min at room temperature to remove any remaining percoll. Washed cells were subjected to qPCR and flow cytometry methods to check purity of isolated microglial cells.
Total RNA isolation, real-time quantitative PCR and absolute quantification by digital PCR Total RNAs were extracted from brain tissues (hippocampus) by using TRI Reagent ® (TR 118) (Molecular Research Center, Inc., Cincinati, OH, USA). To measure mRNA expression, cDNA was synthesized using RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific) followed by qPCR using 5 × HOT FIREPol ® EvaGreen ® qPCR Supermix (Solis BioDyne, Tartu, Estonia) on a QuantStudio 12KFlex instrument (Thermo Fisher Scientific) according to the instructions of the respective manufacturers. Primer sequences for target genes were given in the Supplement Table 1. Absolute quantification of microRNAs was carried out according to the manufacturer's instructions (Qiagen) using miRCURY LNA RT Kit (Cat. No. 339340) for cDNA synthesis and miRCURY LNA PCR Assays (Assay IDs: YP00204688, YP02119310) in combination with QI Acuity EG PCR Kit (Cat. No. 250111) for detection of miR-146a and miR-146b. The samples were run and analyzed on QIAcuity One dPCR System using QIAcuity 26k Nanoplates (all from Qiagen). Relative miRNA expression was measured using TaqMan ® MicroRNA Assays hsa-miR-146a (Assay ID: 000468, Life technologies) and TaqMan ® MicroRNA Assays hsa-miR-146b (Assay ID: 001097, Life technologies, Carlsbad, CA, USA) according to the manufacturer's instructions. For cRNA synthesis, TaqMan ® MicroRNA reverse transcription kit (4366596, Thermo Scientific) and for qPCR, 5× HOT FIREPol ® Probe qPCR Mix Plus (ROX) (Solis BioDyne) were used, respectively. U6 snRNA (Assay ID: 001973, Life Technologies) was used for the normalization of RT-qPCR. To analyze relative mRNA expression, DDCt calculations was used. As housekeeping genes for normalization, Gapdh was used. The data were analyzed in relative to the mean value of one of the samples as a calibrator (control sample or WT saline), which was normalized to 1 and the rest of the samples were compared against the control sample.
For NF-kB p65 protein in the nuclear fraction was performed as follows. Nuclear extract was prepared from the hippocampal tissues according to manufacturer's protocol (Abcam nuclear extraction kit, ab113474) using 20 mg tissue. Protein content of nuclear fractions were measured using Bradford reagent (Sigma, B6916). Equivalent amounts of proteins were resolved on 10% polyacrylamide gels by SDS-PAGE. Resolved proteins were transferred to Immobilon ® FL membrane (IPFL00010, Millipore) in 0.1 M Tris-base, 0.192 M glycine and 20% (w⁄w) methanol using an electrophoretic transfer system with cold-block. The membranes were blocked using Odyssey blocking buffer (LICOR Biosciences, 927-40000) at room temperature for 1 h. After blocking, the membranes were incubated sequentially overnight with different primary antibodies anti rabbit NF-kB p65 (Abcam antibody, ab 16502), anti-rabbit histone-4 antibody to mark the nuclear fraction (Abcam antibody, ab 10158), anti-mouse beta-tubulin antibody to show no cytoplasmic contamination in nuclear fractions (Milipore, MAB1637). Incubations were followed by 3 times washing in Tris Buffered Saline containing 0.1% Tween-20 followed by incubation with the appropriate secondary antibody (1:10000), either goat anti-rabbit IRDye 800CW or 680LT or goat anti-mouse IRDye 680LT (all from Licor Biosciences), for 2 h at room temperature. Immuno-reactive bands were detected by the Odyssey Infrared Imaging System. Densitometry analysis of protein bands were done by Image studio Ver 2.0. Ink.

Statistical analysis
GraphPad 8.0.1 (San Diego, CA, United States) was used for statistical analyses and graphical presentations. The following steps of statistical evaluation of the data were undertaken. All data sets were tested for homogeneity of variances using the Brown-Forsythe test and for normal distribution using the Shapiro-Wilk test. If the data sets passes both the tests then they were processed using two-way ANOVA followed by Tukey multiple comparisons test. If data sets did not pass a test for homogeneity of variances, they were processed using Welch's ANOVA followed by Dunnett's test. If the data did not pass the Shapiro-Wilk test, they were transformed into logarithms (Y=Log(Y)) and processed further using one-way ANOVA followed by Tukey multiple comparisons test. The appropriate statistical test for each data set is specified in the figure legends. Statistical significance was set at p < 0.05, and data were reported as mean ± SEM.

Results
Expression profile of miR-146a/b upon LPS in the hippocampus and microglial cells isolated from the mouse brain To better understand the importance of miR-146a/b in neuroinflammation, we first measured the expression levels of miR-146a/b in the hippocampus of WT mice upon LPS treatment using RT-qPCR method that enables absolute quantification. In hippocampal tissues, basal levels of miR-146a/b were similar, and after LPS administration both miR-146a (p = 0.0013) and miR-146b (p = 0.0170) were significantly induced at 24 h time point. The upregulation of miR-146a was apparently stronger, compared to miR-146b (p = 0.0836) ( Figure 2A). Next, we evaluated expression of miR-146a/b in microglial cells isolated from the WT mouse brain. Purity of isolated microglia is shown in Supplement Figure 1. In microglial cells, the basal expression of miR-146a was several folds higher than that of miR-146b, while upon LPS challenge, both miR-146a (p = 0.0340) and miR-146b (p = 0.0342) were upregulated ( Figure 2B). Significant effects of LPS treatment (F Altogether, these data demonstrate that miR-146a was over 37-fold more highly expressed in microglial cells under basal conditions, and that both miR-146a/b were induced upon LPS treatment in the hippocampus and microglial cells of mouse brain. In contrast, the level of induction of miR-146b was little more prominent than miR-146a in microglial cells.

LPS-induced reduced sickness behavior in Mir146b-/mice
We have previously demonstrated that Mir146b-/-mice did not differ from WT animals with regard of body weight, locomotor activity, food and water consumption, hippocampal and total brain volumes at the age of 2-3 months (22). It is known that systemic administration of LPS in animals leads a behavioral state called "sickness behavior", which is characterized by lethargy, decreased locomotor activity and appetite, depression-like behavior, and increased sensitivity to pain (39). To evaluate the impact of miR-146b on the LPS-induced sickness behavior, we treated Mir146b-/and WT mice with LPS and assessed the locomotor activity, loss of body weight and immobility time in tail suspension test.
We first evaluated changes in body weight before and 24 h after a high dose administration of LPS (1.5 mg/kg) and observed that LPS induced a decrease in body weight in both WT and Mir146b-/-mice (Welch's ANOVA (F(3,40.12) = 120.3, p < 0.0001). Dunnett's test revealed a significant effect (p < 0.001) of LPS treatment in both WT and Mir146b-/-mice. However, LPS-induced body weight loss was slightly less prominent in Mir146b-/-mice than that observed in WT mice (p = 0.07) ( Figure 3A).
Furthermore, we evaluated the development of depressionlike behavior as evidenced by immobility time in the tail suspension test and observed increased immobility time upon LPS treatment (Welch's ANOVA demonstrated a highly significant effect of LPS (F(3,23.1)=17.19, p < 0.0001). Post hoc analysis revealed a significant enhancement of immobility in WT mice upon LPS challenge (p < 0.0001). In contrast, no enhanced immobility was observed in Mir146b-/-mice compared to the untreated group ( Figure 3C).
In conclusion, LPS-induced sickness behavior manifested by the rapid decrease in body weight, reduction in locomotor activity and development of depression-like behavior was less severe in Mir146b-/-mice as compared with WT littermates.

LPS induced less activation in microglial morphology and Iba1 protein levels in
Mir146b-/mice LPS is known to act through the innate immune toll-like 4 (TLR4) to induce an innate immune response, which causes, among other symptoms, sickness behavior. TLR4 is highly expressed in brain microglia, and excessive inflammation resulting from activation of this pathway in the brain has been implicated in various brain pathologies (40). Therefore, we next evaluated LPS-induced microglial activation by assessing their profiles of morphological changes. To do that, we stained the brain sections from WT and Mir146b-/-mice with antibody against Iba1, a marker for microglia ( Figure 4A) and assessed microglial morphometrics, such as average cell size, cell body size and size of dendritic processes.
Our results demonstrated that LPS administration induced trends of increase in microglial density in both WT and Mir146b-/-mice ( Figure 4B). Significant effect of LPS treatment (F(1,17) = 13.18, p = 0.0021) but not genotype and interaction were observed.
Next, we assessed microglial cell size ( Figure 4C), cell body size ( Figure 4D), and size of the dendritic processes ( Figure 4E) separately. We detected increased values of all these parameters upon LPS challenge in both WT and Mir146b-/-mice, but which were less pronounced in Mir146b-/-mice compared to WT group. For average cell size, significant effects existed for LPS treatment (F(1,17)   In addition, we measured LPS-induced changes in Iba1 protein levels in the hippocampus of Mir146b-/-and WT littermates ( Figures 4F, G). For Iba1 protein levels Welch's ANOVA demonstrated significant effect (F(3,5.1) = 10.58, p = 0.028). Post hoc analysis demonstrated a significant increase in Iba1 protein upon LPS challenge in WT animals, but no s i gn ifi c a n t c ha n g e s we r e o b s e r v e d i n M i r 1 4 6 b -/mice ( Figure 4G).
These results together demonstrated that LPS-induced activation of microglia, manifested by increased cell size, cell body size and size of the dendritic processes, was less pronounced in Mir146b-/-mice as compared to WT mice. To assess the activation level of microglial cells after LPS stimulation, percentage of major histocompatibility complex II (MHC-II)-positive M1-type of pro-inflammatory microglia and mean fluorescence intensity (MFI) level of CD11b were measured. CD11b is constitutively expressed by microglia, but its expression is increased upon activation (41)(42)(43). The percentage of MHCII expressing microglia were significantly higher in WT LPS group (p = 0.0142) and Mir146b-/-LPS (p = 0.0131) compared to their respective controls ( Figure 5E). Significance of LPS treatment (F(1,22) = 22.69, p < 0.0001) but not genotype and interaction were observed. As expected, the MFI level of CD11b was significantly higher after administration of LPS in both WT (p < 0.0001) and Mir146b-/-mice (p < 0.0001). No significant difference between the WT LPS and Mir146b-/-LPS groups was detected. However, the basal level of CD11b was lower in Mir146b-/-mice compared to WT mice (p = 0.0056) ( Figure 5F), indicating reduced basal activation of Mir146b-/-microglia. Statistical analysis revealed significant effect of LPS treatment (F(1,22) = 132.2, p < 0.0001), genotype (F(1,22) = 9.717, p = 0.0050), and interaction (F(1,22) = 5.700, p = 0.0260). Altogether, these results suggest that microglial cells in Mir146b-/-mice showed less activation as compared to WT mice.

LPS induced changes in microglial profile in Mir146b-/mice
We also investigated the expression of macrophage colonystimulating factor 1 receptor, Csf1r (CD115) on microglial surface. CSF1R signaling is essential for microglial survival, proliferation, and differentiation (44)(45)(46)(47). Altered expression of CSF1R in microglia have been identified in many neuroinflammatory diseases (48,49). An in vivo study had shown that inhibition of CSF1R attenuated neuroinflammation and reduced microglial proliferation in a murine acute LPS model (50). We found that LPS induced an increase in (p = 0.0411) Csf1r expression in the hippocampus of WT mice but decreased its expression in Mir146b-/-mice (p < 0.0001). Notably, the basal level of Csf1r was higher in Mir146b-/-mice compared to WT mice (p < 0.0001) ( Figure 5G). Statistical analysis showed significant effects of genotype (F(1,22) = 4.535, p = 0.0446) and interaction (F(1,22) = 36.05, p < 0.0001) but not LPS treatment. Altogether, flow cytometry analysis results indicate that microglia of miR-146b deficient mice were less activated and more resistant to LPS treatment.

LPS induced changes in microgliamediated phagocytic activity in
Mir146b-/mice LPS-induced neuroinflammation has been linked to enhanced phagocytosis (51). Phagocytosis is an event, which has been associated with an anti-inflammatory profile of activated microglia (52). Therefore, we evaluated the microglial phagocytic activity upon LPS challenge in Mir146b-/-mice using intracellular staining Vglut2, a glutamatergic pre-synapse-specific protein, by flow cytometry similarly as performed before in (34,53). Gating strategy with representative dot plots are presented in ( Figure 6A). Statistical analysis revealed that Mir146b-/-mice have significantly higher percentage of Vglut2+ microglia than WT mice (p = 0.0062) in basal conditions suggesting that miR-146b deficiency results in an increased phagocytic activity of microglia. Upon LPS treatment, we observed higher levels of Vglut2 engulfment in WT mice (p = 0.0029), whereas Mir146b-/-mice showed a decrease in percentage of Vglut2+ microglia (p = 0.0033) ( Figure 6B) compared to their controls. Statistical analysis revealed no significant effect of LPS treatment (F(1,22) = 0.57, p = 0.46) and genotype (F(1,22) = 0.23, p = 0.63) and highly significant effect of interaction (LPS x genotype, F(1,22) 24.59, p < 0.0001). These results together suggest that miR-146b deficiency reduces microglial ability to be converted into phagocytic phenotype upon LPS treatment.

LPS induced changes in cytokine gene expression in Mir146b-/mice
To answer the question whether reduced ability of LPS to induce neuroinflammation in Mir146b-/-mice was associated with changes in mediators of neuroinflammation, we evaluated mRNA expressions of genes encoding pro-inflammatory cytokines (Il1b, Il18, Tnf, Il6, and ccl5), anti-inflammatory cytokines (Il10 and Il13), and NLRP3 inflammasome component (Nlrp3). Under basal conditions, the mRNA expression levels of these genes did not significantly differ between WT and Mir146b-/-mice. However, the marked differences between genotypes appeared upon LPS administration (Figure 7).

miR-146a is upregulated in the hippocampus and microglial cells isolated from miR-146b deficient mice
To answer the question of what mechanisms might be responsible for the observed attenuation of LPS-induced neuroinflammation in Mir146b-/-mice we measured the expression of miR-146a in these animals. First, we measured absolute miR-146a levels in the hippocampal tissues and secondly, we measured relative expression of miR-146a in microglial cells isolated from whole brain using percoll gradient. Experiments demonstrated significantly higher expression of miR-146a upon LPS challenge in the hippocampal tissue of Mir146b-/-mice (p < 0.0001) as compared to WT animals (p = 0.0016) ( Figure 8A). A significant effect of LPS treatment (F(1,12) = 106.0, p < 0.0001) but not genotype and interaction (LPS x genotype, F(1,12) = 10.74, p = 0.0066) was found. In isolated microglial cells, both WT and Mir146b-/mice showed overexpression of miR-146a upon LPS administration. Welch's ANOVA of miR-146a expression in microglial cells also demonstrated a significant effect on LPS (F (3,4.2) = 86.89, p = 0.0003), whereas post hoc analysis showed revealed much higher increase (p = 0.0006) in miR-146a upon LPS challenge in Mir146b-/-mice as compared with WT mice. It should be also noted that basal miR-146a expression in Mir146b-/-mice was also significantly (p = 0.001) higher than in WT mice ( Figure 8B). Gene expressional changes in the hippocampus of Mir146b-/-mice reveal reduced inflammatory response to LPS. Total RNA was purified from hippocampus of mice injected with LPS in saline or equal volume of saline (control).
Il10 and (F) Il13 was analyzed by RT-qPCR with primers for indicated genes. Number of animals = 7. Data represented as mean ± SEM; * p < 0.05, ** p < 0.01, **** p < 0.0001 (Data in panels (A-C, E, F) were analyzed using two-way ANOVA with Tukey's multiple comparisons test; data in panel (D) did not show normal distribution and were transformed into logarithms (Y=log(Y) and further processed using one-way ANOVA with Tukey's multiple comparisons test).

Mir-146a/b-/mice
To evaluate whether miR-146a upregulation is responsible for the observed attenuation of neuroinflammation in Mir146b-/-mice, additional experiments were performed on Mir-146a/b-/-mice. We assessed LPS-induced sickness behavior in these animals and measured the mRNA expression of proinflammatory cytokines Ccl5 and Tnf in the hippocampal tissues. In Mir-146a/b-/-mice, LPS administration induced sickness behavior similar to WT mice. For body weight changes, Welch's ANOVA showed significant effect LPS (F(3,15.3) = 76.2, p < 0.0001), whereas post hoc analysis showed a similar decrease in body weight in both WT and Mir-146a/b-/-mice ( Figure 9A). For locomotor activity Welch's ANOVA also showed a significance (F(3, 8.759 = 21.26, p = 0.002) and post hoc analysis demonstrated significant decrease in locomotion in WT (p < 0.05) and Mir-146a/b-/-mice (p = 0.01) ( Figure 9B). Immobility time in tail suspension test was significantly (p = 0.05) increased in Mir-146a/b-/-mice and a trend toward increase was also seen in WT animals ( Figure 9C). The lack of significance in WT animals is probably due to the insufficient number of animals in a group. Further gene expression analysis in Mir-146a/b-/-mice showed an increased expression of proinflammatory cytokines upon LPS challenge (Figures 9D, E). For Tnf expression, Welch's ANOVA showed significant effect (F LPS induced NF-kB activation, Irak1 expression and interferon regulatory factor 7 (IRF7) expression in Mir146b-/mice To explore further mechanisms of the observed LPS-induced upregulation of miR-146a in Mir146b-/-mice, we focused on the transcriptional regulators of miR-146a expression NF-kB and Irf7/3.
As the expression levels of cytokines are NF-kB-dependent and NF-kB is the most important transcriptional regulator of miR-146a/b, we next assessed whether miR-146b deficiency may affect this pathway. The classical NF-kB pathway activation occurs via phosphorylation and following ubiquitination and degradation of IkB. The remaining NF-kB dimer (e.g., p65/p50 subunits) translocate to the nucleus, where it binds to the DNA consensus sequence of various target genes. Measuring p65 subunit in the nuclear fraction reflects the level of activation of the NF-kB pathway (54). Therefore, we next isolated the nuclear fraction of hippocampal cells and measured p65 and histone-4 levels with western blot. In WT mice, LPS treatment induced significant NF-kB activation (p = 0.039) as evidenced by the increased levels of p65 in the nuclear lysate. In contrast, no significant increase in p65 levels was observed in nuclear lysates purified from Mir146b-/-mice upon LPS challenge (Figures 10A,  B) and the whole blot picture is provided in the (Supplement Figure 4). For p65 levels, two-way ANOVA demonstrated significant effect of LPS treatment (F(1,10) = 15.3; p = 0.0029) and significant effect of interaction between treatment and genotype (F(1,10) = 4.98; p = 0.05) but no effect of genotype.
We next analyzed mRNA expression of a miR-146a/b target, Irak1 and TLR4 receptor protein levels in the hippocampus of WT and Mir146b-/-mice after LPS treatment. Interestingly, mRNA expression of Irak1 was reduced in both WT and Mir146b-/-mice in response to LPS. However, the extent of Irak1 reduction was more prominent in WT (p < 0.0001) than Mir146b-/-mice (p = 0.0478) ( Figure 10C). Significant effects of LPS treatment (F(1, 24) = 34.30, P < 0.0001) and genotype (F(1, 24) = 4.421, P = 0.0462), but not interaction were observed. As previous data have demonstrated that upregulated miR-146a can negatively affect TLR4 expression (55)(56)(57), we analyzed TLR4 expression in miR-146b deficient mice upon LPS administration. Lysates of hippocampal tissues probed for TLR4 demonstrated a significant increase in TLR4 protein levels upon LPS administration in WT animals but not in Mir146b-/-mice ( Figures 10E, F). These data suggest that miR-146a-mediated ability of targeting TLR4 might be responsible for the observed attenuation of NF-kB activation in Mir146b-/-mice. Next, we explored a possible role of another group of transcriptional regulators Irf3/7. For Irf3, two-way ANOVA did not demonstrate significant effect. Multiple comparisons test revealed a small but significant increase in expression upon LPS treatment only in Mir146b-/-mice (Supplement Figure 5). For Irf7, two-way ANOVA demonstrated highly significant effect of LPS treatment: (F(2,23) = 82.4, p < 0001), significant effect of genotype: (F(2,23) = 11.9, p = 0.002), and significant effect of interaction: (F (2,23) = p = 0.02). Multiple comparisons test showed a significant increase in Irf7 expression upon LPS challenge in both WT (p < 0.001) and Mir146b-/-(p < 0.0001) mice ( Figure 10D). Furthermore, LPS-induced changes in Irf7 expression were significantly (p < 0.001) higher in Mir146b-/-mice as compared with their WT littermates. These data suggest that Irf7 is most probably responsible for the observed upregulation of miR-146a in Mir146b-/-mice.

Discussion
Previous research has demonstrated that miR-146 family members act as negative modulators of inflammation (12,28,31). Several other studies have also demonstrated that deficiency in miR-146a results in an exaggerated inflammatory response to the LPS challenge (18,19,58), whereas miR-146a overexpression reduces microglia mediated neuroinflammatory responses and inhibits sickness behavior (59,60). Paradoxically, our study revealed that Mir146b-/-mice demonstrated attenuated neuroinflammatory response upon LPS challenge as compared with their WT littermates. Reduced neuroinflammatory status of Mir146b-/-mice upon LPS challenge was evidenced by the reduced intensity of sickness behavior and demonstrated less morphological characteristics of microglial activation. Although sickness behavior is believed to be associated with neuroinflammation, the impact of peripheral inflammatory reaction cannot be excluded (61, 62). Concordantly, flow cytometry experiments demonstrated an increased percentage of microglia in WT mice but not in Mir146b-/-mice upon LPS administration. In addition, the activation marker for microglial CD11b was decreased at basal levels in Mir146b-/-mice indicating that miR-146b deficiency causes lesser microglial activation. Furthermore, LPS mediated increased phagocytosis capacity in WT microglia and but not in Mir146b-/-mice.
In line with behavior studies and microglial status, LPSmediated stimulation of the expression of pro-inflammatory cytokines Il1b, Ccl5, Tnf, Il18, Il6 and Nlrp3 mRNAs was weaker in Mir146b-/-LPS mice compared to WT LPS animals. These data indicate that miR-146b deficient mice are more immunotolerant to the LPS-induced inflammatory signal than their WT littermates.
One possible explanation for a seemingly paradoxical consequence of miR-146b deficiency may come from the observed over-expression of miR-146a in these animals when treated with LPS. The increase in expression of miR-146a upon LPS challenge was evident in hippocampal tissues as well as in microglia isolated from the hippocampal tissue. Although the only marginal increase in miR-146a copy number in these animals was observed, the relevance of miR-146a over-expression in Mir146b-/-mice was confirmed by the experiments conducted on double knockout mice deficient for both miR-146a and miR-146b. Indeed, in these animals, LPS-induced sickness behavior and expression of pro-inflammatory cytokines were similar or even higher than those observed in WT animals.
It seems that blunted inflammatory response upon LPS challenge observed in Mir146b-/-mice is due to the reduced activation of NF-kB. The mechanisms of miR-146a-mediated reduction of neuroinflammatory response remains obscure. It seems unlikely that major regulatory activator of NF-kB, Irak1 is implicated in the observed reduced activity of NF-kB as reduction in the expression of Irak1 was higher in Mir146b-/mice as compared with WT animals. More likely, reduced activation of NF-kB is due to the diminished ability of TLR4 to become upregulated in response to LPS. Indeed, several studies have demonstrated upregulation of TLR4 protein upon LPS challenge (63,64). In our experiments, similar upregulation was also observed in WT animals and in contrast, Mir146b-/mice TLR4 protein remained unchanged after LPS administration. It is known that TLR4 is a target of miR-146a (55)(56)(57) and thereby causing decreased activation of NF-kB and reduced inflammation.
To explain possible mechanisms of the observed miR-146a upregulation we next searched for transcription regulators such as interferon regulatory factor 3/7 (Irf3/7) responsible for miR-146a overexpression. There is evidence that Irf3/7 plays an essential role in the LPS-induced interferon-b gene expression and endotoxic shock (65) and overexpression of Irf3/7 suppressed inflammatory reaction upon LPS challenge (66). Indeed, our experiments demonstrated that Irf7 and, to a lesser extent, Irf3 become highly upregulated upon LPS challenge in miR-146b deficient mice. We, therefore, propose that upregulation of Irf7 leads to the over-expression of miR-146a, which in turn inhibits NF-kB activation leading to the blunted inflammatory response. The crosstalk between miR-146a and miR-146b in the regulation of neuroinflammation is summarized in Figure 11.
It should be noted that the mechanism by which miR-146b can regulate the expression of Irf3/7 remains unknown and needs to be studied in the future. In addition, a possible impact of other transcriptional regulators such as CCAAT-enhancer-bindingprotein-b (C/EBPb) in the observed overexpression of miR-146a in Mir146b-/-mice upon LPS treatment remains unknown. We also do not exclude the impact of systemic inflammation and peripheral miR-146b deficiency in the observed blunted neuroinflammatory response in miR-146b deficient mice. It is not excluded that miR-146b deficiency can promote invasion of the peripheral immune cells expressing miR-146a to the brain via disruption of the blood-brainbarrier (BBB). Wnt/b-catenin signaling is essential for maintaining the integrity of the adult BBB in physiological and pathological conditions (67) as its inhibition or deficiency causes BBB breakdown. Previous studies have shown that miR-146b induces silencing of Secreted frizzled-related protein 1 (SFRP1) which acts as an antagonist of Wnt signaling (68). Thus, miR-146b deficiency might induce indirect inhibition of Wnt/b-catenin signaling and weakness of BBB. This hypothesis, however, remains highly speculative as no direct studies on BBB permeability in miR146b deficient mice were conducted so far.

Conclusion
In the present study, we utilized different methods to examine LPS-induced neuroinflammation in miR-146b deficient mice. It was found that LPS-induced neuroinflammation is attenuated in these animals and it was evidenced by the reduced signs of sickness behavior, and reduced activation of microglia. Gene and protein expression analysis revealed that LPS administration induced weaker upregulation of proinflammatory cytokines and NF-kB activation in the brain of Mir146b-/-mice compared to WT mice. The blunted neuroinflammatory reaction upon LPS challenge observed in Mir146b-/-mice is the most probably due to the compensatory over-expression of miR-146a. Over-expression of miR-146a is mediated by the induction of transcription regulator Irf7 and, to a lesser extent, Irf3 in the hippocampal tissue. Our data show the existence of crosstalk between miR-146a and miR146b in the regulation of neuroinflammation.

Data availability statement
The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding author.

Ethics statement
The animal study was reviewed and approved by Animal Experimentation Committee at the Estonian Ministry of Agriculture (no. 183 and 158, 2021).   Summary of the crosstalk between miR-146a and miR.146b in the regulation of neuroinflammation. miR-146b leads to over-expression of miR-146a transcriptional regulator Irf7, which in turn induces upregulation of miR-146a. miR-146a inhibits activation of TLR4 upon LPS challenge and consequent inhibition of neuroinflammation. Red arrows show inhibition; blue arrows show activation.


An open-access data set of pig skin anatomy and physiology for modelling purposes

Abstract The use of animal as opposed to human skin for in vitro permeation testing (IVPT) is an alternative, which can reduce logistical and economic issues. However, this surrogate also has ethical considerations and may not provide an accurate estimation of dermal absorption in humans due to physiological differences. The current project aimed to provide a detailed repository for the anatomical and physiological parameters of porcine skin, with the aim of parametrizing the Multi-phase Multi-layer Mechanistic Dermal Absorption (MPML MechDermA) Model in the Simcyp Simulator. The MPML MechDermA Model is a physiologically based pharmacokinetic (PBPK) model that accounts for the physiology and geometry of skin in a mechanistic mathematical modelling framework. The database provided herein contains information on 14 parameters related to porcine skin anatomy and physiology, namely, skin surface pH, number of stratum corneum (SC) layers, SC thickness, corneocyte thickness, corneocyte dimensions (length and width), volume fraction of water in corneocyte (where SC is divided into four parts with different water contents), intercellular lipid thickness, viable epidermis thickness, dermis thickness, hair follicle and hair shaft diameter, hair follicle depth and hair follicle density. The collected parameters can be used to parameterize PBPK models, which could be further utilized to bridge the gap between animal and human studies with interspecies extrapolation or to predict dermatokinetic properties typically assessed in IVPT experiments. Database URL: https://data.mendeley.com/datasets/mwz9xv4cpd/1

Background
When evaluating dermal absorption either in vivo or ex vivo, the gold standard would usually be to conduct a study with human patients or with excised human skin, respectively. However, this is not always possible due to logistical, economical or ethical limitations. An alternative approach is to conduct studies on animals or use excised animal skin; however, data applicability and interpretation can be challenging due to physiological differences between the animal of choice and human.
In vitro permeation testing (IVPT) is a widely used method for evaluating cutaneous permeability of topical drug products (1,2) and dermal exposure assessment (3)(4)(5). Excised adult human skin is recommended for that purpose, and the results from such experiments have been shown to correlate well with in vivo data (6). The skin for these studies is commonly obtained from cosmetic surgery procedures or cadavers and stored frozen. However, the supply of human skin can often be low and expensive. In addition, each piece of skin is relatively small, usually only allowing a maximum of 6-8 diffusion cells per donor. Storage conditions might influence skin integrity, viability or enzyme activity and thus observed dermal permeation (7-10).
As an alternative, animal skin models are often used as a tool to estimate dermal absorption. The use of animal skin affords many advantages such as being easier to obtain, ability to more easily use fresh skin, and, in the case of larger animals such as pig, a large number of cells can be set up from one donor, resulting in reduced interindividual variability for investigative studies (11)(12)(13)(14). Porcine skin is often used as it can be easily obtained from animals slaughtered for food and is most similar to human skin in morphological structure and immunohistochemical properties (15)(16)(17)(18).
Despite their similarities, permeation of some topically applied compounds can vary significantly between porcine and human skin (19,20); the extent of this difference depends on the nature of the compound and formulation in which it is applied. Moreover, since even in vitro studies of topical drugs are associated with ethical issues, credible alternative models that can help informed decision during pharmaceutical formulation development are highly beneficial.
Physiologically based pharmacokinetic (PBPK) modelling accounts for physiology and geometry of the skin in a mechanistic modelling framework (21). One powerful feature of PBPK models is the ability to extrapolate between systems, such as from healthy individuals to a diseased population (22)(23)(24), adult to paediatric (25)(26)(27) or animal to human (28)(29)(30). To accurately extrapolate between two systems, the physiological differences between them must be well characterized. For this purpose, the current work aimed to collate a database of anatomical and physiological data describing the skin of the domestic and miniature pig. The provided data set may be utilized for the development of PBPK models. The current work aimed specifically to parameterize the in vitro module of the Multi-phase Multi-layer Mechanistic Dermal Absorption (MPML MechDermA) Model (IVPT module) in the Simcyp Simulator ® (Certara, Princeton, NJ, USA) for porcine skin. However, since the database consists of parameters that describe skin anatomy, it can also be utilized to parametrize other PBPK dermal models such as the Transdermal Compartmental Absorption and Transit model in the GastroPlus ® (Simulations Plus, Inc.) and any other models, which require such input (31).
The MPML MechDermA Model accounts for the physiology of the skin by incorporating a database of physiological data describing the geometry and structure of each layer (21). Currently, the model database incorporates nine body sites of human skin.

Data collection
The premise for the presented database was to provide a detailed repository for the anatomical and physiological parameters of the porcine skin. The literature search encompassed 20 parameters that describe skin in the MPML Mech-DermA Model (21). Six of these were not included in the final data set, namely, corneocyte pH, lipid and sebum viscosity, density of lipids and proteins and tortuosity, due to a lack of quantitative data in the literature. Moreover, skin temperature was not searched because under the in vitro conditions, this is a parameter of the experiment, not a physiological value.
All data were extracted manually from the relevant papers. Whenever numerical values were not specified in the text, but the microscopic picture together with the scale bar was given or data were presented on the chart, they were obtained using the ImageJ 1.53k and GetData Graph Digitizer 2.26 software, respectively. Units were unified. In one paper, the given units were assumed to be incorrect due to an editorial error, so they were changed from millimetres (mm) to the micrometres (μm), and the information about this was added to the 'comments' column (32). Whenever the standard error (SE) was given instead of standard deviation (SD), but there was sample size (n) reported, the SD value was calculated. Furthermore, coefficient variation was calculated when the SD value was available. Parameters were split by the body site (such as ear, abdomen and back), and if there was any detailed information on the specific part of the body site (such as the inner or outer part of the ear), it was also included in the body site column. In the MPML Model for the fraction of water in corneocytes, the stratum corneum (SC) layers were divided into four equal-sized bins. Therefore, in the current work, the water content for the whole SC was expressed as mean values for 0-25%, 25-50%, 50-75% and 75-100% of the depth, for the top, middle 1, middle 2 and bottom layers, respectively. The SC thickness for each calculation was taken from the specific paper. In some papers, the viable epidermis (VE) thickness was not given, but it was possible to calculate by subtracting the SC thickness from whole epidermis thickness. A similar approach was taken to extract the corneocyte thickness, where it was calculated as the thickness of SC divided by the number of layers of SC, with the respect of intercellular lipid thickness sourced from the study by Charalambopoulou et al. (33).

Database structure
The database is provided in the form of a Microsoft Excel spreadsheet. It contains information on 14 parameters related to the porcine skin anatomy and physiology, i.e. skin surface pH, number of SC layers, SC thickness, corneocyte thickness, corneocyte dimensions (length and width), volume fraction of water in corneocyte (where SC is divided into four parts with different water contents), intercellular lipid thickness, VE thickness, dermis thickness, hair follicle and hair shaft diameter, hair follicle depth and hair follicle density. Each parameter was described using characteristics shown in Table 1. Missing data were left blank in the data set; additional important information can be found in the 'comments' column.

Database utilization
The database consists of porcine skin parameters, which may influence drug penetration, permeation and absorption, specifically those required to parameterize the in vitro module of the MPML MechDermA Model. Therefore, the data can be used to generate an ex vivo porcine skin model, which enables ether predictions of dermatokinetic properties, or perform interspecies extrapolation. However, the model will need to be verified for this purpose. Furthermore, the database contains information about the type of skin (ex vivo/in vivo/fresh/frozen) and which specific porcine skin location was used, as well as the type of pig (pig or minipig), species and age, so it may be utilized to analyse the influence of those physiological subsets on the outcome of dermal absorption experiments.

Database limitations
Relevant parameters were extracted manually; however, due to the lack of consistency in nomenclature and methods, there is some uncertainty. Anatomically, epidermis is a part of the skin that is divided into the SC and VE. However, often, in the literature, a sample of VE is referred as the epidermis. Whenever the word 'epidermis' was used, and no procedure that would suggest that by this word, authors meant 'viable epidermis' only was mentioned in the Methods section, the 'viable epidermis' thickness was calculated as the difference between the total thickness of 'epidermis' and the 'stratum corneum' thickness if both were provided. Moreover, there was some inconsistency in the definition of 'rete pegs' in the VE. The dermoepidermal junction is undulating, and it is possible to distinguish the 'thinner part' (without rete pegs) and 'thick part' (with rete pegs). However, the authors did not always state which part had been included in their measurements of 'viable epidermis thickness'. Whenever such information was available, this is written in the 'comments' column.

Database access
The data set of pig skin anatomy and physiology is freely available under the open-access license at https://data.mendeley. com/datasets/mwz9xv4cpd/1 and as a Supplementary file.


Piper tectoniifolium Kunth: A New Natural Source of the Bioactive Neolignan (−)-Grandisin

The Piper species are a recognized botanical source of a broad structural diversity of lignans and its derivatives. For the first time, Piper tectoniifolium Kunth is presented as a promising natural source of the bioactive (−)-grandisin. Phytochemical analyses of extracts from its leaves, branches and inflorescences showed the presence of the target compound in large amounts, with leaf extracts found to contain up to 52.78% in its composition. A new HPLC-DAD-UV method was developed and validated to be selective for the identification of (−)-grandisin being sensitive, linear, precise, exact, robust and with a recovery above 90%. The absolute configuration of the molecule was determined by X-ray diffraction. Despite the identification of several enantiomers in plant extracts, the major isolated substance was characterized to be the (−)-grandisin enantiomer. In vascular reactivity tests, it was shown that the grandisin purified from botanical extracts presented an endothelium-dependent vasorelaxant effect with an IC50 of 9.8 ± 1.22 μM and around 80% relaxation at 30 μM. These results suggest that P. tectoniifolium has the potential to serve as a renewable source of grandisin on a large scale and the potential to serve as template for development of new drugs for vascular diseases with emphasis on disorders related to endothelial disfunction.

Introduction
The plant kingdom is a natural complex library of bioactive molecules with a range of therapeutic potential. As such, it is in high demand in the pharmaceutical industry as a valuable source for novel drug leads and discovery [1]. Lignans represent a class of natural products derived from the oxidative coupling of two C 6 -C 3 units, which have their origin in the shikimic acid pathway and cinnamic acid derivatives [2]. Most lignans are found in a free form in nature that can be isolated from different parts of a plant. Among these are lignans and neolignans composed of phenolic compounds that present a wide spectrum of biological activities [3,4]. Despite their wide distribution in vascular plant species, their biological functions in plants are still not fully determined [5]. A recognized natural source of many bioactive lignan and neolignan derivatives is the Piper species [3,6]. In Brazil numerous medicinal plants rich in lignan metabolites, including the Piper species, are used in alternative folk medicine for a number of ailments and physical conditions. For example, ethnopharmacological studies on the traditional use of Piper truncatum Vell suggested a potential use in vascular diseases that has led to the isolation and characterization of potentially active lignans with biomedical applications for hypertension [7].
A recent trend has been the investigation of plants containing the neolignan grandisin, which is a tetrahydrofuran (THF) lignan trimethoxy substituted metabolite shown to present remarkable biological activities. Phytochemical investigations described the isolation and characterization of this natural compound as one of the major secondary metabolites from the bark of Cryptocarya crassinervia Miq. (Lauraceaea) [8], Piper solmsianum [9] and Virola surinamensis (Rol. exRottb), Myrtaceace [10]. The most commonly isolated grandisin enantiomer is the (−)-grandisin form that is an all-trans THF lignan. However, many other enantiomers, such as (+)-grandisin, may also be present in lignan pools due to the presence of multiple chiral centers. An example is the isolation of (+)-grandisin from Piper polysyphorum, a native species from the south of China [11].
It is well known that the use of plant-derived compounds as a starting point for the development of new therapeutics is particularly challenging due to the quantities and purity necessary to permit in vitro screenings and in vivo trials. Too frequently, a target compound can only be isolated in small quantities in a laborious separation process from complex crude extracts that requires multiple steps and makes it difficult to continuously support biological assays [12]. Yet, multiple published results related to (−)-grandisin have shown a wide variety of pharmacological properties such as antitumoral [13][14][15], larvicidal [16], antimalarial [17], antinociceptive and anti-inflammatory [10], trypanocidal [9], and leishmanicidal [18] as well as being a competitive inhibitor of CYP2C9, CYP3A4/5 and the CYP450 enzyme type [19]. In view of the potential pharmacological value of this natural metabolite, it is important to identify and characterize natural sources that can meet the demands of in vitro and in vivo investigations as well as the industrial requirements for product development and fabrication. Here, we report on the potential of P. tectoniifolium Kunth, a native Brazilian shrub in the southeast region, to serve as a renewable botanical source of (−)-grandisin. A new HPLC-based method was developed to accompany and evaluate purified material. In addition, the absolute configuration of (−)-grandisin was determined and its pharmacological performance was confirmed.

Chemical Characterization of Piper tectoniifolium as a Source of (−)-Grandisin
The present study is the first report on an HPLC validated method for the quantification of the neolignan grandisin from the species P. tectoniifolium. This species is an evergreen shrub of medium size that is native to Brazil, which is presented as a renewable botanical source for the preparative isolation of the tetrahydrofuran neolignan in sufficient quantities for industrial applications. Phytochemical studies led to the characterization of this metabolite as the major compound in extracts of leaves, branches, and inflorescences. From nonpolar extracts (n-hexane), separation procedures led to the isolation of a large amount of (−)-grandisin from leaves (968.0 mg from 8.2 g of n-hexane extract; 11.8%) and from branches (180.0 mg from 1.0 g of n-hexane extract; 18.0%). The results confirm the bioprospecting potential of the P. tectoniifolium species as a source for this bioactive neolignan.
The HPLC-DAD-UV method developed was validated and proved to be selective for the identification of (−)-grandisin as well as sensitive, linear, precise, exact, robust and with a recovery level above 90%. The grandisin signal in the new method showed a high retention factor (α = 5.0) and signal symmetry (~1.0). No interference was observed in the chromatographic window for (−)-grandisin, even in high sample concentration ( Figure 1). results confirm the bioprospecting potential of the P. tectoniifolium species as a source for this bioactive neolignan.
The HPLC-DAD-UV method developed was validated and proved to be selective for the identification of (−)-grandisin as well as sensitive, linear, precise, exact, robust and with a recovery level above 90%. The grandisin signal in the new method showed a high retention factor (α = 5.0) and signal symmetry (~1.0). No interference was observed in the chromatographic window for (−)-grandisin, even in high sample concentration ( Figure 1). The linearity of the analytical method was determined from analyses performed on three different days over the concentration range of 5, 10, 20, 30, 50, 80, 100 and 200 μg/mL, the r > 0.99 (r = 1.00000 ± 5.77 × 10 5 ) without outliners and with a homoscedastic distribution. The formula to calculate (−)-grandisin in the samples was ABS (mAU) = (31,678 ± 127) concentration (μg/mL) + (11,621 ± 1344) considering the three analytical curves (Supplementary Materials, Figure S1). The variation between intraday and interday values showed that the method was precise since the RSD values were less than 15% (Supplementary Materials, Table S1). Accuracy was tested for all concentrations in the analytical curve and demonstrated a minimum value of 96.4% for 5 μg/mL and a maximum of 102.5% for 30 μg/mL. These values suggest that the method is accurate based on an allowed variance between 85 to 115% (Supplementary Materials, Table S2). For values of small parameters variation, the method proved to be robust, using up to five different changes. The parameter that demonstrated the greatest amplitude in the percentage variation coefficient was the pH (2.42% for 150 μg/mL and 1.46% for 15 μg/mL). However, the percentage variation coefficient for quantification purposes was less than 5% and therefore acceptable (Supplementary Materials, Tables S3 and S4). The analysis of standards at concentrations of 30 μg/mL and 60 μg/mL showed high recovery values of above 90% (Supplementary Materials, Table S5). The limit of detection (LOD) and quantification (LOQ), obtained by successive dilution was registered at 75 and 100 ng/mL, respectively, which are compatible with UV detection (10 −9 ).
Overall, the new HPLC method can be considered validated to be sensitive for the detection and quantification of (−)-grandisin. For comparison, the published LOD and The linearity of the analytical method was determined from analyses performed on three different days over the concentration range of 5, 10, 20, 30, 50, 80, 100 and 200 µg/mL, the r > 0.99 (r = 1.00000 ± 5.77 × 10 5 ) without outliners and with a homoscedastic distribution. The formula to calculate (−)-grandisin in the samples was ABS (mAU) = (31,678 ± 127) concentration (µg/mL) + (11,621 ± 1344) considering the three analytical curves (Supplementary Materials, Figure S1). The variation between intraday and interday values showed that the method was precise since the RSD values were less than 15% (Supplementary Materials, Table S1). Accuracy was tested for all concentrations in the analytical curve and demonstrated a minimum value of 96.4% for 5 µg/mL and a maximum of 102.5% for 30 µg/mL. These values suggest that the method is accurate based on an allowed variance between 85 to 115% (Supplementary Materials, Table S2). For values of small parameters variation, the method proved to be robust, using up to five different changes. The parameter that demonstrated the greatest amplitude in the percentage variation coefficient was the pH (2.42% for 150 µg/mL and 1.46% for 15 µg/mL). However, the percentage variation coefficient for quantification purposes was less than 5% and therefore acceptable (Supplementary Materials, Tables S3 and S4). The analysis of standards at concentrations of 30 µg/mL and 60 µg/mL showed high recovery values of above 90% (Supplementary Materials, Table S5). The limit of detection (LOD) and quantification (LOQ), obtained by successive dilution was registered at 75 and 100 ng/mL, respectively, which are compatible with UV detection (10 −9 ).
Overall, the new HPLC method can be considered validated to be sensitive for the detection and quantification of (−)-grandisin. For comparison, the published LOD and LOQ to quantify benzofuran neolignans in extracts of Piper regnelli were higher and in the range of 1.68 and 5.60 µg/mL, respectively [20]. Since grandisin is considered as a potential antitumoral drug candidate, previous studies have investigated the concentration of grandisin obtained from Piper solmisianum and from Virola surinamensis in biological fluids and nanoformulations, respectively [21,22]. In biological fluids, grandisin and four other metabolites were quantified with an initial retention time (Rt) of 15 min, which was optimized to 6.0 min of Rt, although the method used to determine (−)-grandisin metabolism was validated for the analysis of drugs. In the second investigation, PLGA nanocapsule formulations loaded with grandisin were evaluated with regard to the release of grandisin to improve its solubility. Despite the short Rt for grandisin, this method was not validated. The method presented here proved to be selective and displayed good resolution for grandisin characterization in complex natural matrices as crude extracts from plants. The quantification of the crude extracts of P. tectoniifolium with the newly developed and validated method are shown in Table 1. The extraction of leaves with n-hexane transitioned a large quantity of grandisin (527.80 mg/g), which demonstrated its affinity for solvents with a low polarity. The results suggest P. tectoniifolium nonpolar extracts could serve as valuable sources for the isolation of (−)-grandisin on a large scale. However, the extraction from leaves should be considered since it would represent a more ecofriendly natural source to obtain the metabolite. The high quantities of (−)-grandisin in the inflorescences may be related to a chemical defense system, a hypothesis that would need to be investigated. In a comparison between methanol extracts, the amount of (−)-grandisin in leaves was 17.70 mg/g, which was less than in inflorescences (20.80 mg/g) and branches (78.40 mg/g). Again, considering an industrial procedure to obtain (−)-grandisin: while the branches showed a higher concentration with methanol, the collection of branches can lead to plant exhaustion and could make maintaining production more difficult compared to leaves. According to our results, the concentration of grandisin in the leaves and bark of P. tectoniifolium was higher than the published values for Cryptocarya crassinervia Miq. (Lauraceaea) [8], Piper solmsianum [9] and Virola surinamensis (Rol. exRottb), Myrtaceace [10] species.

Crystalline and Molecular Structure of (−)-Grandisin Confirmed by X-ray Diffractometry
The dihedral angle formed between the two benzene rings was verified to be 120 • by X-ray diffractometry ( Figure 2A). The data suggested that the crystalline structure is formed from interactions of π bonds in positions 1 and 1 with hydrogen atoms of the methoxy groups in positions 4 and 4 ( Figure 2B). The structure of (−)-grandisin was determined as showed in Figure 2C.
However, through a critical analysis of the chromatograms and mass spectra obtained from different samples, it was possible to detect the occurrence of other substances with the same molecular weight, but in lower concentrations that are most likely grandisin isomers. This conclusion would agree with the variety of THF neolignans derived from grandisin such as all cis and all trans THF lignans found by Ramos et al. (2017), which were also found in Piper solmsianum. The confirmation of the corrected configuration among aryls and methyl groups presented in THF lignans such as grandisin is fundamental to expanding the understanding on stereochemical diversity and its possible effects on pharmacological assays [23]. However, through a critical analysis of the chromatograms and mass spectra obtained from different samples, it was possible to detect the occurrence of other substances with the same molecular weight, but in lower concentrations that are most likely grandisin isomers. This conclusion would agree with the variety of THF neolignans derived from grandisin such as all cis and all trans THF lignans found by Ramos et al. (2017), which were also found in Piper solmsianum. The confirmation of the corrected configuration among aryls and methyl groups presented in THF lignans such as grandisin is fundamental to expanding the understanding on stereochemical diversity and its possible effects on pharmacological assays [23].

Pharmacological Evaluation
The previous chemical and pharmacological investigations of P. tectoniifolium suggested that this species was the source of an THF neolignan endowed with a vasodilating effect that still lacks studies to clarify the mechanisms of action. To examine the pharmacological activities of (−)-grandisin purified from P. tectoniifolium, rat aorta rings were prepared and precontracted with 1 μM phenylephrine (PHE). The The perspective of the asymmetric part of the molecular structure of (−)-grandisin with complete molecular structure with numbering scheme and displacement ellipsoids drawn at the 50% probability level; (B) perspective of the electronic interactions between (−)-grandisin molecules for the formation of the crystalline reticulum (perspective view of the C-H···π interactions (dashed cyan lines) dimer in grandisin, with graph-set R 4 4 (12)); (C) absolute configuration of grandisin determined by X-ray (2R,3S,4S,5R)-3,4-dimethyl-2,5-bis(3,4,5trimethoxycyclohexyl)oxolane.

Pharmacological Evaluation
The previous chemical and pharmacological investigations of P. tectoniifolium suggested that this species was the source of an THF neolignan endowed with a vasodilating effect that still lacks studies to clarify the mechanisms of action. To examine the pharmacological activities of (−)-grandisin purified from P. tectoniifolium, rat aorta rings were prepared and precontracted with 1 µM phenylephrine (PHE). The concentration-dependent relaxation (IC 50 = 9.8 ± 1.22) effect of (−)-grandisin on aorta arterial rings with their endothelium intact is shown in Figure 3 (closed boxes). It was observed that at a concentration of 30 µM,~80% relaxation was measured. Removal of the vascular endothelium considerably abolished the relaxation caused by (−)-grandisin (Figure 3, closed circles), which suggests that the vascular effect of (−)-grandisin acts on endothelium cells. arterial rings with their endothelium intact is shown in Figure 3 (closed boxes). It was observed that at a concentration of 30 μM, ~80% relaxation was measured. Removal of the vascular endothelium considerably abolished the relaxation caused by (−)-grandisin ( Figure 3, closed circles), which suggests that the vascular effect of (−)-grandisin acts on endothelium cells. Studies with THF lignans from other Piper species such as (−)-cubebin isolated from P. cubeba L. have also demonstrated a vasorelaxant effect. However, the effect appeared to be irrespective of the presence of endothelium. Carvalho et al. (2013) showed that (−)cubebin was able to reduce PHE contraction in intact rings, suggesting that the THF lignan promotes vasorelaxation via the NO/cGMP pathway in rat aorta without prostacyclin involvement [24]. In another study, Raimundo et al. (2009) demonstrated the vasodilatory activity of crude extracts of leaves of P. truncatum Vell. as well as the pure isolated lignan eudesmin, suggesting the possible therapeutical properties of these natural products for the treatment of hypertension according to traditional medicine. In this case, nitric oxide release and the involvement of the histamine receptor in the endothelial cells were related to the observed biological effects [7].
Endothelial dysfunction is one of the most relevant features in the pathophysiology of hypertensive disorders such as hypertension. Substances that directly affect endothelial functional regulation such as (−)-grandisin studied in the present paper may have significant potential in the treatment of circulatory diseases [25]. Plant-derived natural products can be useful for drug discovery development, especially when they are based on previous traditional knowledge. However, bioprospection from botanicals can be very challenging, depending on plant species and the concentration of biological markers. Since plant sources are often slow growing and tend to accumulate target compounds at very low quantities over long growth periods or in specific climate conditions, it is of great importance to identify natural sources that can supply the scale needed to meet market demands [12]. Our results from extracts of different parts of P. tectoniifolium Kunth strongly suggest that this plant could be a renewable source to produce (−)-grandisin on a large scale that could allow the development of new treatments for hypertension. Studies with THF lignans from other Piper species such as (−)-cubebin isolated from P. cubeba L. have also demonstrated a vasorelaxant effect. However, the effect appeared to be irrespective of the presence of endothelium. Carvalho et al. (2013) showed that (−)cubebin was able to reduce PHE contraction in intact rings, suggesting that the THF lignan promotes vasorelaxation via the NO/cGMP pathway in rat aorta without prostacyclin involvement [24]. In another study, Raimundo et al. (2009) demonstrated the vasodilatory activity of crude extracts of leaves of P. truncatum Vell. as well as the pure isolated lignan eudesmin, suggesting the possible therapeutical properties of these natural products for the treatment of hypertension according to traditional medicine. In this case, nitric oxide release and the involvement of the histamine receptor in the endothelial cells were related to the observed biological effects [7].
Endothelial dysfunction is one of the most relevant features in the pathophysiology of hypertensive disorders such as hypertension. Substances that directly affect endothelial functional regulation such as (−)-grandisin studied in the present paper may have significant potential in the treatment of circulatory diseases [25]. Plant-derived natural products can be useful for drug discovery development, especially when they are based on previous traditional knowledge. However, bioprospection from botanicals can be very challenging, depending on plant species and the concentration of biological markers. Since plant sources are often slow growing and tend to accumulate target compounds at very low quantities over long growth periods or in specific climate conditions, it is of great importance to identify natural sources that can supply the scale needed to meet market demands [12]. Our results from extracts of different parts of P. tectoniifolium Kunth strongly suggest that this plant could be a renewable source to produce (−)-grandisin on a large scale that could allow the development of new treatments for hypertension.

Chemicals
All solvents, i.e., technical grade n-hexane, chloroform, methanol, acetonitrile and HPLC grade acetonitrile and methanol were provided by TEDIA, Brazil. MilliQ deionized water was obtained at the laboratory and acidified with glacial acetic acid (Merck, Brazil).

Extract Preparation and Separation Procedures
Dried plant material consisting of branches (260 g), leaves (232 g) and inflorescences (85 g) were maintained separate and reduced to small fragments. The plant materials were separately extracted by static maceration with n-hexane, methanol over three days with daily solvent exchange.

Separation Procedures (−)-Grandisin Isolation by Precipitation
A portion of an n-hexane extract obtained from branches was prepared to be submitted to countercurrent chromatography separation (CCC). The chromatographic profile chosen was composed of a mixture of n-hexane (Hex)/ethyl acetate (EtOAc)/MeOH/H 2 O at a 4:2:2:0.5 ratio. When the sample (1.0 g) was introduced into the chosen solvent system, a precipitate of white crystals in the bottom of the flask was observed ( Figure 4A). To favor further precipitation in a slow and uniform manner, the flask was covered with a watch glass. After 24 h, the precipitate was observed to form needle-like crystals ( Figure 4B). The remaining solvent was removed leaving only semipurified crystals yielding 180 mg.
All solvents, i.e., technical grade n-hexane, chloroform, methanol, acetonitrile and HPLC grade acetonitrile and methanol were provided by TEDIA, Brazil. MilliQ deionized water was obtained at the laboratory and acidified with glacial acetic acid (Merck, Brazil).

Botanical Material
The

Extract Preparation and Separation Procedures
Dried plant material consisting of branches (260 g), leaves (232 g) and inflorescences (85 g) were maintained separate and reduced to small fragments. The plant materials were separately extracted by static maceration with n-hexane, methanol over three days with daily solvent exchange.

Separation Procedures (−)-Grandisin Isolation by Precipitation
A portion of an n-hexane extract obtained from branches was prepared to be submitted to countercurrent chromatography separation (CCC). The chromatographic profile chosen was composed of a mixture of n-hexane (Hex)/ethyl acetate (EtOAc)/MeOH/H2O at a 4:2:2:0.5 ratio. When the sample (1.0 g) was introduced into the chosen solvent system, a precipitate of white crystals in the bottom of the flask was observed ( Figure 4A). To favor further precipitation in a slow and uniform manner, the flask was covered with a watch glass. After 24 h, the precipitate was observed to form needle-like crystals ( Figure 4B). The remaining solvent was removed leaving only semipurified crystals yielding 180 mg.

(−)-Grandisin Isolation by Silica Gel Chromatographic Column
Adsorption chromatography on a silica gel column was utilized to process an nhexane extract from leaves of P. tectoniifolium (8.2 g). The components of the extract were eluted from the column by solvent systems prepared with binary mixtures of Hex, EtOAc and MeOH in a gradient of increasing polarities that resulted in 63 fractions. Based on similar TLC profiles, fractions 39-50 that eluted from the column in concentrations ranging from 20-50% (n-Hex/EtOAc) were pooled (2.0 g). The pooled fractions were subjected to a second fractionation process also using a silica gel column that yielded 120 fractions. Through the comparison with the chromatographic standard previously obtained, it was possible to confirm by TLC presence of grandisin in fractions 41-63. The fractions enriched with grandisin were next subjected to exclusion chromatography on a Sephadex LH-20 column using a 3:1 mixture of methanol/chloroform. This procedure led to a yield of 968.0 mg of grandisin with relative purity above 94.0% (analyzed by GC-FID and GC-MS).

GC-FID Analysis
Quantitative and qualitative analyses were performed on a GC 2010 Shimadzu equipped with a ZB-1MS fused silica capillary column (30 m × 0.25 mm i.d. × 0.25 µM film thickness). The operating temperatures were 260 • C at the injector, 290 • C at the detector and from 60 • C to 290 • C at 10 • C/min in a column oven. Hydrogen was used as the carrier gas at a constant flow of 1.0 mL/min. The percent purity of the compound was obtained from the peak areas of the GC-FID analysis.

GC-MS Analysis
Qualitative analyses were performed on a GC-QP2010 PLUS Shimadzu equipped with a ZB-5MS fused silica capillary column (30 m × 0.25 mm i.d. × 0.25 µM film thickness). The operating temperatures were injector, 270 • C; detector, 290 • C; and from 60 • C up to 290 • C at 3 • C/min in the column oven. Helium (99.99%) was used as the carrier gas at a constant flow at 1.0 mL/min for GC coupled to mass spectrometer (MS) analysis. The target compound was identified by comparison of its mass spectra (see Supplementary Materials) with published data [9].

NMR Analysis
Nuclear magnetic resonance spectra of hydrogen and carbon ( 13 C and 1 H NMR) were obtained on Varian device VNMRS-Gemini 500 spectrometer operating at a frequency of 400 MHz ( 1 H)/100 MHz ( 13 C) using CD 3 OD as the solvent. Special techniques and bidimensional measurements such as COSY, HMBC, HSQC were also performed. The chemical shift values (δ) in dimensionless units were referred to an internal standard (TMS), being represented in parts per million (ppm) of the applied frequency for each experiment and coupling constants (J) were measured in Hz. The obtained data (see Supplementary Materials) were compared to literature data [9].

Quantification of the Major Isolated Lignan (−)-Grandisin from Extracts of P. tectoniifolium
The quantification of (−)-grandisin was performed at the Laboratory of Environmental Toxicology of the Oswaldo Cruz Foundation. For the quantification of (−)-grandisin, a Shimadzu ® CLASS-VP High-Performance Liquid Chromatograph coupled to a Diode Array Ultraviolet detector (HPLC-DAD-UV) was used. Additional equipment included a controller (SCL 10A VP), degasser (DGU-14A), binary pump (LC-10AD VP), oven (CTO-10AS VP) and detection system (DAD SPD M10A VP). Chromatograms were analyzed using the Schimadzu Class VP ® program version 6.1. All runs were performed with a Thermo ODS Hypersil column (250 mm × 4.6 mm i.d. × 5 µM, particle size) using a mobile phase in the isocratic mode with a 65/35 mixture of acetonitrile/MilliQ deionized water acidified with glacial acetic acid (pH 3.0) at a flow rate of 1.0 mL/min and an oven temperature at 50 • C for detection at 234 nm. The injector washing solvent was methanol. The total analysis time was 8 min with an average pressure of 76 bar. Under these conditions, the retention time (rt) of (−)-grandisin was registered to be 6.6-7.0 min. Standard and sample injection volume was 20 mL.
This original method was developed and validated for the analysis of grandisin in extracts of P. tectoniifolium in relation to selectivity, linearity, precision, accuracy, limit of detection (LOD) and quantification (LOQ), robustness and recovery in accordance with DOQ-CGCRE-008-V2 from the National Standards for Analytical Validation of The National Institute of Metrology, Standardization and Industrial Quality/Brazil [26]. A stock solution of grandisin in methanol at 200 mg/mL was prepared on the day of the analysis to generate solutions of 5, 10, 20, 30, 50, 80 and 100 µg/mL. Linearity was determined from Molecules 2022, 27, 1151 9 of 11 these concentrations over three different days. Accuracy was determined to represent the degree of agreement between the determinations obtained by the method under study in relation to a value accepted as a reference and which was also tested for those concentrations. Precision was expressed as the standard deviation (SD) or relative standard deviation (RSD) from measurements of 15 and 150 µg/mL obtained on three different days (inter-day) as well as morning and afternoon runs (intraday). The robustness of an analytical method is a measure of its ability to resist small and deliberate variations in the analytical parameters, being indicative of reliability during normal use of the instrument. This parameter was tested in relation to acetonitrile in the mobile phase, flow rate, pH, temperature and acid type. Recovery measures the efficiency of the extraction procedure for an analytical method. Recovery tests were performed by spiking 30 or 60 µg/mL of grandisin standard solution into the samples. LOD and LOQ were obtained by serial dilution and analysis of the relation of signal to noise that is 1/3 and 1/10, respectively. All analysis was performed in triplicate from solution of the extracts and fractions from P. tectoniifolium.

X-ray Diffractometry
A single crystal grandisin was analyzed by X-ray diffraction to determine the absolute configuration of the molecule, considering that it has four stereogenic centers. Data collections was in a KappaCCD Nonius diffractometer, Mo radiation, crystal system hexagonal, space group P6522. The structure was solved by direct methods and revealed that in the grandisin, [C 24 H 32 O 7 ], the asymmetric unit is composed by half molecule with the oxygen O1 in a special position, on a rotation axis, n = 2, and the symmetry operation {x, −y, −z} gives rise to the complete molecule. After refinement the absolute configuration of the molecule was confirmed by the low value of the Flack parameter.

Pharmacological Evaluation
Animals were anesthetized by isoflurane (1% mg/kg) inhalation, and after the loss of motor control, rats were euthanized by cervical dislocation. Subsequently, the thoracic aorta was dissected and cleansed of connective tissue to obtain rings of 2 mm in length that were suspended in a 5 mL organ bath (myograph model 620M, DMT, Denmark). Tissues were kept at 37 • C in a Krebs Henseleit buffer (120 mM NaCl, 5 mM KCl, 1.1 mM MgCl 2 , 2.5 mM CaCl 2 , 1.2 mM NaH 2 PO 4 , 10 mM N- [2-hydroxyethyl] piperazine-N'-[2-ethanesulfonic acid] (HEPES), 15 mM NaHCO 3 and 11 mM glucose; pH 7.4) that was constantly bubbled with a gas mixture of 95% O 2 and 5% CO 2 . Each arterial segment was suspended between two steel hooks connected to an isometric transducer to measure tension through a data acquisition system (PowerLab 8 and LabChart Pro, AD-Instruments, Australia). After a stabilization period of 60 min at a rest tension of 1.0 g with periodic changes of solution (every 15 min), a stable contraction was achieved with 1 µM phenylephrine (PHE). Functional endothelial integrity was assessed by the ability of 10 µM acetylcholine to induce ≥70% relaxation in vessels precontracted with 1 µM PHE [27]. For vascular reactivity tests, the rings were preconstricted with 1 µM of PHE and after a stable response, the concentrations (µM) of 0.3, 1.0, 3.0, 10.0 and 30.0 of (−)-grandisin were subsequently added to the chamber and the level of relaxation measured. Values were expressed as mean ± standard error of mean (SEM). The IC 50 values (defined as the concentration of the test compound that reduced 50% of the maximal contraction) were obtained by an actual concentration-response curve fitting using GraphPad Prism 6.0 software (GraphPad Software, San Diego, CA, USA).

Ethics
All animal studies (male Wistar rats (200-300 g) were carried out in strict accordance with the guidelines approved by the Animal Use Ethics Committee from the Federal University of Rio de Janeiro (CEUA/CCS/UFRJ 087/15).

Conclusions
For the first time, the evergreen shrub species P. tectoniifolium has been presented as a natural alternative source for grandisin, a bioactive metabolite with several pharmacological activities with an emphasis toward disorders related to endothelial disfunction. The medicinal interest for this neloginan in drug discovery strategies led to our development of a new HPLC method that was validated for its analysis in plant extracts. The method was also useful for quality control and to obtain fingerprints of various botanical medicines. The highest concentration of the target compound was identified in nonpolar extracts of leaves (≈53%), which is a suitable amount for mass production. Considering that it is a renewable source, it would be of interest to conduct further research on phytochemical bioprospection strategies to enhance the yields of secondary metabolites such as (−)-grandisin from nonpolar leaf extracts of P. tectoniifolium. Together, the results suggest that the botanical source, methods and yields have the potential to provide the metabolite for development of new drugs for vascular diseases.